Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 5'-flanking DNA of the mouse RII beta subunit of the cAMP-dependent protein kinase gene was characterized by transient transfection of RII beta-CAT constructs into mouse neuroblastoma cells (NB2a) and Chinese hamster ovary (CHO) cells and by gel mobility shift and footprinting assays. The minimal promoter of the RII beta gene was composed of two adjacent functional elements. A 3'-element which supported enhanced CAT activity was located between base pairs (bp) -267/-168 from the translation initiation start site. CAT plasmids containing these RII beta sequences showed 12- and 16-fold increased CAT activity in the NB2a and CHO cells, respectively, compared to the basic CAT vector. Plasmids containing 20 additional bp 5' to the -267/-168 fragment showed 2-fold more CAT activity than the shorter fragment in NB2a cells, while CAT activity in CHO cells was nearly the same for both constructs. CAT plasmids containing only this 20-bp fragment showed 9- and 13-fold increased CAT activity in NB2a and CHO cells, respectively. The core promoter of the RII beta gene lacked classical TATA and CAT sequences, but contained 3 copies of the Sp1 core consensus sequence. Gel mobility shift assays using 32P-labeled 5'-flanking DNA containing bp -291/-49 and nuclear extracts from NB2a and CHO cells displayed several retarded bands in the gels suggesting complex formation with nuclear DNA-binding factors. Unlabeled DNA containing bp -291/-49 blocked the appearance of all retarded bands. Competition using an oligonucleotide corresponding to the Sp1 DNA-binding site effectively blocked the appearance of the two more slowly migrating bands but did not affect the major rapidly migrating bands. DNase I footprinting analysis using purified Sp1 protein confirmed that Sp1 could bind to the Sp1 sites. Methylation interference and mutational analysis showed that one of the faster migrating bands was the result of factor binding to the DNA sequence adjacent to the Sp1 sites. Additional tissue-specific nuclear-binding factor sequences were detected upstream of the core promoter. Our data suggest that the core promoter of the RII beta gene can initiate transcription from the DNA around the Sp1 sites but that there are tissue-specific nuclear factor-binding sites located distal to the Sp1 sites.
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PMID:Characterization of a minimal promoter element required for transcription of the mouse type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase. 133 64

Previous studies showed that the core promoter of the mouse cAMP-dependent protein kinase regulatory subunit type II beta (RII beta) gene was composed of two functional elements. One element was GC rich and bound the Sp1 transcription factor. The second element contained a helix-loop-helix (HLH)-motif. Each element conferred transcriptional activity when inserted upstream of a reporter gene, chloramphenicol acetyltransferase and transfected into mouse NB2a neuroblastoma cells and Chinese hamster ovary (CHO) cells. The core promoter was further characterized by mutational analysis using electrophoretic mobility shift assays and by transfection into CHO and NB2a cells. Electrophoretic mobility shift assays showed that the HLH-consensus motif, CACGTG, present in the RII beta gene bound nuclear factors present in NB2a and CHO cells. Mutations in the HLH-core motif decreased the binding of these factors and reduced the transcriptional activity of constructs containing the chloramphenicol acetyltransferase reporter when transfected into these cells. The results showed that the central nucleotides as well as the adjacent bases were important for the interaction with the nuclear binding factors. UV cross-linking, Southwestern blot analysis, and interference of the mobility shift patterns by specific antisera directed against USF and c-Myc indicated that both of these transcription factors were forming complexes with the HLH-consensus motif. The results suggest that RII beta transcription may be regulated, in part, by USF and c-Myc in NB2a and CHO cells.
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PMID:Association of USF and c-Myc with a helix-loop-helix-consensus motif in the core promoter of the murine type II beta regulatory subunit gene of cyclic adenosine 3', 5'-monophosphate-dependent protein kinase. 783 49

The rat lactate dehydrogenase (LDH) A subunit gene promoter contains a putative AP-1 binding site at -295/-289 bp, two consensus Sp1 binding sites at -141/-136 bp and -103/-98 bp, and a single copy of a consensus cyclic AMP-responsive element (CRE) at -48 to -41 bp upstream of the transcription initiation site. Additionally, an as yet unidentified silencer element is located within the -1173/-830 bp 5'-flanking region. Transient transfection analyses of a -1173/+25 bp LDH A-chLoramphenicol acetyltransferase fusion gene has indicated a complete inability of the promoter fragment to direct basal or forskolin-induced transcription. Deletion of the -1173/-830 bp sequence restored basal and cyclic AMP (cAMP)-inducible activity. Point mutations in the Sp1 binding sites of a -830/+25 bp promoter fragment reduced basal but not the relative degree of cAMP-inducible activity. cAMP-regulated transcriptional activity was dependent upon an 8 bp CRE, -TGACGTCA-, located at the -48/-41 bp upstream region. Mutations in the CRE abolished cAMP-mediated induction and reduced basal activity by about 65%. The CRE binds a 47 kDa protein which has previously been identified as CRE binding protein (CREB)-327, an isoform of the activating transcription factor/CREB transcription factor gene family. Co-transfection of a vector that expresses the catalytic subunit of cAMP-dependent protein kinase stimulates LDH A subunit promoter activity suggesting that cAMP induces LDH A subunit gene expression through phosphorylative modification of CREB-327. This study emphasizes a fundamental role of several modules including Sp1 and CREB binding sites in regulating basal and cAMP-mediated transcriptional activity of the LDH A gene.
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PMID:Analysis of the rat lactate dehydrogenase A subunit gene promoter/regulatory region. 799 73

We have characterized three cis-acting elements of the human CYP11A1 gene. A proximal cAMP-responsive sequence (P-CRS) functioned in both adrenal Y1 and placental JEG-3 cells. An upstream cAMP-responsive sequence (U-CRS) and an enhancer, localized by transfections of deleted gene segments linked to a reporter gene to bases -1621 to -1503 and -1931 to -1822, respectively, functioned in Y1 but not JEG-3 cells. Both regions bind proteins only from Y1 cells as identified by footprinting analysis. U-CRS contains the TCAAGGTCA sequence that binds the nuclear receptor family of proteins. The cAMP-dependent transcription mediated by U-CRS, but not by P-CRS, was abolished in a cell line deficient in cAMP-dependent protein kinase. Therefore, P-CRS and U-CRS use different effectors to mediate cAMP response. Gel mobility shift, competition, and antibody supershift experiments showed that nucleotides -117 to -94, which contributed to P-CRS activity in transfection experiments, bound weakly to Sp1-like proteins. This feature is shared by many proximal regulatory elements of steroidogenic genes. Therefore, steroidogenic genes could be coordinately regulated through common regulatory elements such as P-CRS, U-CRS, and cell type-selective enhancers.
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PMID:Actions of two different cAMP-responsive sequences and an enhancer of the human CYP11A1 (P450scc) gene in adrenal Y1 and placental JEG-3 cells. 811 86

Neural-specific expression of the mouse regulatory type-I beta (RI beta) subunit gene of cAMP-dependent protein kinase is controlled by a fragment of genomic DNA comprised of a TATA-less promoter flanked by 1.5 kilobases of 5'-upstream sequence and a 1.8-kilobase intron. This DNA contains a complex arrangement of transcription factor binding motifs, and previous experiments have shown that many of these are recognized by proteins found in brain nuclear extract. To identify sequences critical for RI beta expression in functional neurons, we performed a deletion analysis in transgenic mice. Evidence is presented that the GC-rich proximal promoter is responsible for cell type-specific expression in vivo because RI beta DNA containing as little as 17 base pairs (bp) of 5'-upstream sequence was functional in mouse brain. One likely regulatory element coincides with the start of transcription and includes an EGR-1 motif and 3 consecutive SP1 sites within a 21-bp interval. Maximal RI beta promoter activity required the adjacent 663 bp of 5'-upstream DNA where most, but not all, of the regulatory activity was localized between position -663 and -333. A 37-bp direct repeat lies within this region that contains 2 basic helix-loop-helix binding sites, each of which are overlapped by two steroid hormone receptor half-sites, and a shared AP1 consensus sequence. Intron I sequences were also tested, and deletion of a 388-bp region containing numerous Sp1-like sequences lowered transgene activity significantly. These results have identified specific regions of the RI beta promoter that are required for the expression of this signal transduction protein in mouse neurons.
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PMID:Promoter sequences in the RI beta subunit gene of cAMP-dependent protein kinase required for transgene expression in mouse brain. 857 64

Transcription factor Sp1 is a phosphoprotein whose level and DNA binding activity are markedly increased in doxorubicin-resistant HL-60 (HL-60/AR) leukemia cells. The trans-activating and DNA binding properties of Sp1 in HL-60/AR cells are stimulated by cAMP-dependent protein kinase (PKA) and PKA agonists and inhibited by PKA antagonists as well as by the PKA regulatory subunit. Reporter gene activity under the control of the Sp1-dependent SV40 promoter is stimulated in insect cells transiently expressing Sp1 and PKA, and the DNA binding activity of recombinant Sp1 is activated by exogenous PKA in vitro. These results indicate that Sp1 is a cAMP-responsive transcription factor and that Sp1-dependent genes may be modulated through a cAMP-dependent signaling pathway.
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PMID:Modulation of transcription factor Sp1 by cAMP-dependent protein kinase. 926 Nov 18

The expression of multidrug-resistance (MDR) in breast carcinoma cell line MCF-7/ADR50 is primarily dependent on the transcriptional activation of the MDR1 gene. We now report that MDR in this cell line is partially reversed by the type I cAMP-dependent protein kinase (PKA) inhibitor, 8-Cl-cAMP. MDR1 promoter activity was also regulated through a PKA-dependent pathway and was inhibited by 8-Cl-cAMP, and stimulated by the enantiomeric agonist, SpcAMP[S]. MDR1 promoter activity through an Sp1 response element was stimulated by exogenous Sp1, a factor that we have shown to be activated by PKA. These results indicate that MDR1 promoter activity is linked to the cAMP/PKA signaling pathway, and that PKA antagonists may be useful for reversing the multidrug-resistant phenotype.
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PMID:Regulation of the MDR1 promoter by cyclic AMP-dependent protein kinase and transcription factor Sp1. 945 66

Uncoupling protein (UCP) 2 is a member of the uncoupling-protein family, and it appears to function as an uncoupler of oxidative phosphorylation. To identify cis-acting regulatory elements controlling this gene's expression, we cloned an approx. 6.2-kb region upstream from the translation-initiation site of the mouse UCP2 gene and analysed its transcription activity using chimaeric mouse UCP2 promoter-placental-alkaline-phosphatase (PLAP) reporter-gene constructs. Sequence analysis showed that the 5'-flanking region of the mouse UCP2 gene was not similar to those of mouse UCP1 or UCP3. For the mouse UCP2, the region near the transcription-initiation site lacked the typical TATA box, but was GC-rich, resulting in presence of several potential specificity protein 1 (Sp-1), activator protein (AP)-1 and AP-2 binding sites. The putative regulatory motifs for muscle-regulatory protein (MyoD), brown-fat regulatory element, CCAAT box, cAMP-response element and Y box were also found in the mouse UCP2 promoter region by computer-assisted analysis. From the results of Northern-blot analysis and transient expression assay, we found that the mouse UCP2 gene responded to the cAMP-dependent protein kinase alpha-catalytic subunit signal activation at the transcription level. Additionally, deletion analysis of the UCP2 promoter-PLAP constructs indicated that the minimal region exhibiting the promoter activity was located between nt -33 and +100, and that a strong enhancer was present within 601 bp of the 5'-promoter region. In particular, the region from nt -233 to -34 significantly induced PLAP activity in the cell lines derived from various tissues and in the primary culture cells of rat brown adipose tissue, suggesting that this region is most important for the ubiquitous expression of mouse UCP2 mRNA. Furthermore, it was shown that two silencer elements were involved in the mouse UCP2 gene; one was located between nt -2746 and -602, and the other was identified in intron 1. These regions deprived the enhancer of the ability to induce PLAP activity. This study shows a fundamental role for positive and negative cis-acting DNA elements in regulating the basal and cAMP-induced transcription activity of the mouse UCP2 gene.
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PMID:Mechanism of ubiquitous expression of mouse uncoupling protein 2 mRNA: control by cis-acting DNA element in 5'-flanking region. 1033 81

The pituitary peptide hormone ACTH regulates transcription of the cholesterol side chain cleavage cytochrome P450 (CYP11A) gene via cAMP and activation of cAMP-dependent protein kinase. A G-rich sequence element conferring cAMP-dependent regulation has been found to reside within region -118 to -100 of the bovine CYP11A promoter. Previous studies have suggested that it binds a protein antigenically related to the transcription factor Sp1. We now report that the -118/-100 element binds both Sp1 and Sp3, members of the Sp family of transcription factors. We have made use of Drosophila SL2 cells, which lack endogenous Sp factors, to dissect the possible functional roles of Sp1, Sp3, and Sp4. All factors stimulated the activity of cotransfected reporter constructs in which the promoter of the bovine CYP11A gene regulates luciferase expression. Sp3 did not repress Sp1-dependent activation, as has previously been shown for other G-rich promoters. Mutation of the -118/-100 element of CYP11A abolished Sp1-mediated activation of a CYP11A reporter gene in SL2 cells as well as cAMP responsiveness in human H295R cells. Furthermore, cotransfection of SL2 cells with the catalytic subunit of cAMP-dependent protein kinase together with Sp1 and a CYP11A reporter construct enhanced Sp1-dependent activation of the reporter 4.2-fold, demonstrating that Sp1 confers cAMP responsiveness in these cells. Thus, we show that introduction of Sp1 alone in an Sp-negative cell such as SL2 is sufficient to achieve the cAMP-dependent regulation observed using the -118/-100 element of CYP11A in adrenocortical cells.
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PMID:Role of Sp1 in cAMP-dependent transcriptional regulation of the bovine CYP11A gene. 1038 57

Regulation of gene transcription is an incompletely understood function of nitric oxide (NO). Human leukocytes produce increased amounts of tumor necrosis factor alpha (TNF-alpha) in response to NO. This effect is associated with decreases in intracellular cAMP, suggesting that NO might regulate gene transcription through promoter sequences sensitive to cAMP such as cAMP response elements (CRE) and Sp1 binding sites. Here we report that a Sp1 binding site in the TNF-alpha promoter conveys NO responsiveness. Human U937 cells were differentiated for TNF-alpha production with phorbol 12-myristate 13-acetate. NO donors and H89, an inhibitor of cAMP-dependent protein kinase increased, while dibutyryl cAMP (Bt(2)cAMP) decreased TNF-alpha promoter activity. Deletion or mutation of the proximal Sp1 site, but not the CRE site, abolished the activating effects of NO donors and H89. Further, NO- and H89-mediated increases in TNF-alpha promoter activity were associated with decreased Sp1 binding. The insertion of Sp1 sites into a minimal cytomegalovirus promoter conferred NO responsiveness, an effect blocked by Bt(2)cAMP. Mutation of these inserted Sp1 sites prevented this heterologous promoter from responding to NO, H89 and Bt(2)cAMP. These results identify the Sp1 binding site as a promoter motif that allows NO to control gene transcription.
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PMID:A Sp1 binding site of the tumor necrosis factor alpha promoter functions as a nitric oxide response element. 1055 88


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